Detection of anti-IgA antibodies using enzyme immunoassay

An enzyme immunoassay is described for the detection of anti-IgA-antibodies in human serum. The principle is based on the binding of the antibodies to IgA-coated polystyrene tubes and their following reaction with peroxidase conjugated Fc-specific anti-human-IgG.     

Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies

Helicobacter pylori is an important human pathogen. Prophylactic immunization with bacterial antigen plus an adjuvant protects mice against challenge with live H. pylori. Surprisingly, it was found that immunizations of mice already infected with Helicobacter also influenced bacterial colonization. This concept of therapeutic immunization is a novel phenomenon. Because H. pylori lives in the lumen of the stomach, it was initially hypothesized that the protective mechanism would involve induction of secretory IgA. However, work with knockout mice has demonstrated that prophylactic immunization is equally effective in mice deficient in IgA and even in microMT mice lacking B lymphocytes. Currently nothing is known about therapeutic vaccination and the effect of immunizing a host with an ongoing ineffective immune response. To address this, we infected B-cell deficient, microMT mice with H. pylori and therapeutically immunized them four times in 3 weeks with bacterial sonicate and cholera toxin adjuvant. These immunizations significantly reduced colonization by H. pylori. The antibody- negative status of the microMT mice was confirmed by ELISA. Thus, therapeutic immunization stimulates an immune response, which reduces H. pylori infection via a mechanism that is antibody independent. How this is achieved remains to be determined, but may well involve a novel immune mechanism.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.     

Continuous-flow/stopped-flow system using an immunobiosensor for quantification of human serum IgG antibodies to Helicobacter pylori

Conventional methods, such as gastric biopsy, enzyme-linked immunosorbent assay (ELISA), culture, require a long time for the determination of Helicobacter pylori infections. This study reports an amperometric immunoreactor for rapid and sensitive quantification of human serum immunoglobulin G (IgG) antibodies to H. pylori. Antibodies in the serum sample are allowed to react immunologically with the purified H. pylori antigens that are immobilized on a rotating disk. The bound antibodies are quantified by horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to human IgG. HRP in the presence of hydrogen peroxide catalyzes the oxidation of hydroquinone to p-benzoquinone. The electrochemical reduction back to hydroquinone is detected on a glassy carbon electrode surface at -0.15 V. The electrochemical detection can be done within 1 min, and the analysis time does not exceed 30 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.6 and 1.9 U ml-1, respectively. The amperometric immunoreactors showed higher sensitivity and lower time consumed than did the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological, and analytical practices.     

幽门螺杆菌的治疗性疫苗

幽门螺杆菌感染的传统治疗方案面临着耐药性和依从性差的问题,而其治疗性疫苗有着广阔的应用前景.该文综述近年来幽门螺杆菌治疗性疫苗的研究进展,分别阐述幽门螺杆菌的感染免疫机制、主要抗原、佐剂、已有的治疗性疫苗等方面.     

Human serum antibody response against iron-repressible outer membrane proteins of Helicobacter pylori

Abstract In Helicobacter pylori , in vitro iron limitation induces the expression of several iron repressible outer membrane proteins (IROMPs), which are not expressed under normal growth conditions. To substantiate their proposed role in virulence of H. pylori , we determined whether these IROMPs are also expressed in vivo. Therefore, we tested whether sera of patients with H. pylori infection contained antibodies against IROMPs. All sera from 20 H. pylori positive patients showed a clear immune response against a 77 kDa heme-binding IROMP in an immunoblot assay. Antibody responses against the other IROMPs were also found, but with lower frequencies. Serum samples from 18 patients negative for H. pylori infection did not show any immunoreactivity with IROMPs. These results indicate that the IROMPs of H. pylori are immunogenic and are expressed in vivo.     

Detection of Helicobacter pylori in cow's milk

AIMS: To investigate the existence of Helicobacter pylori in cow's milk as one of the foods which most Japanese children eat. METHODS AND RESULTS: Detection of H. pylori was demonstrated by the semi-nested polymerase chain reaction (PCR), a culture method and electron microscopy. Semi-nested PCR demonstrated the ureA gene of H. pylori in 13 of 18 (72.2%) raw milk samples and in 11 of 20 (55%) commercial pasteurized milk samples. Helicobacter pylori binding immunomagnetic beads with H. pylori-specific goat anti-H. pylori antibody was shown by electron microscopy in both raw and pasteurized milk positive for the ureA gene. Helicobacter pylori was cultured in one raw milk sample, whereas it was not cultured in pasteurized milk samples. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility that cow's milk is a transmission vehicle in childhood H. pylori infection, although we failed to confirm the survival of H. pylori in pasteurized milk.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Neutralisation of vacuolating activity of cytotoxin by serum antibodies of Helicobacter pylori infected patients

The study involved 196 H. pylori strains and 196 serum samples taken from the same patients. H. pylori strains were investigated for the production of vacuolating cytotoxin. Antibodies to the vacuolating cytotoxin produced by H. pylori were detected in the sera samples by neutralisation assay (on Intestine 407 cells) and ELISA. Of the 196 H. pylori strains tested, 80 (40.8%) were found to express vacuolating cytotoxic activity. The titres of vacuolating cytotoxic were ranged from 1:2 to 1:128. The vacuolating assay was positive in 37.1% strains isolated from children, and in 50% strains isolated from adults. Cytotoxin-positive H. pylori strains were found more frequently in duodenal ulcer (71%) than in chronic gastritis (35.2%) patients, and this difference was statistically significant p<0.05. Neutralising antibodies to vacuolating cytotoxin were present in 51% and 49% of the serum samples tested by neutralisation and ELISA, respectively. Duodenal ulcer patients had antibodies to vacuolating cytotoxin more frequently (p<0.05) than chronic gastritis patients. Antibodies to cytotoxin were detected in 100% of the serum samples from patients infected by cytotoxic H. pylori strains. This suggests that the presence of anticytotoxic antibodies in the serum samples may be regarded as a sensitive indicator of infection by cytotoxic H. pylori strains.     

Progress in vaccine development against Helicobacter pylori

Based on the very high prevalence of diseases caused by Helicobacter pylori, particularly in the developing world, and the rapid emergence of antibiotic resistance among clinical isolates, there is a strong rationale for an effective vaccine against H. pylori. In this review we describe recent promising candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori, as well as studies to identify immune responses that are related to protection in experimental animals. We also describe identification of different types of immune responses that may be related to protection against symptoms based on comparisons of H. pylori-infected patients with duodenal ulcers or gastric cancer and asymptomatic carriers. We conclude that there is still a strong need to clarify the main protective immune mechanisms against H. pylori as well as to identify a cocktail of strong protective antigens, or recombinant bacterial strains that express such antigens, that could be administered by a regimen that gives rise to effective immune responses in humans.     

Helicobacter pylori defense against oxidative attack

Helicobacter pylori is a microaerophilic, gram-negative pathogen of the human stomach. Despite the chronic active gastritis that develops following colonization, H. pylori is able to persist unharmed in the stomach for decades. Much of the damage caused by gastric inflammation results from the accumulation of reactive oxygen/nitrogen species within the stomach environment, which can induce oxidative damage in a wide range of biological molecules. Without appropriate defenses, this oxidative damage would be able to rapidly kill nearby H. pylori, but the organism employs a range of measures, including antioxidant enzymes, biological repair systems, and inhibitors of oxidant generation, to counter the attack. Despite the variety of measures employed to defend against oxidative injury, these processes are intimately interdependent, and any deficiency within the antioxidant system is generally sufficient to cause substantial impairment of H. pylori viability and persistence. This review provides an overview of the development of oxidative stress during H. pylori gastritis and examines the methods the organism uses to survive the resultant damage.     

Helicobacter pylori antibodies and gastric cancer: a gender-related difference

Helicobacter pylori has been proposed as a causative agent of gastric cancer. The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with gastric cancer. Serum samples were collected from 115 Lithuanian patients with non-cardia gastric cancer and 110 age- and sex-matched controls without cancer. Heat-stable, low-molecular-mass, and outer membrane proteins were used as antigens to analyze serum IgG antibody response against H. pylori by enzyme-linked immunosorbent assay. Seroprevalence of H. pylori using low-molecular-mass antigen was significantly higher in gastric cancer patients, compared to controls (77% versus 57%, p<0.05). Significant differences in the prevalence of H. pylori infection between gastric cancer patients and controls were found in females using all three studied antigens: heat-stable (98% versus 84%, p<0.05), low-molecular-mass (88% versus 48%, p<0.05) and outer membrane proteins (78% versus 57%, p<0.05). In males, no significant differences were revealed between gastric cancer patients and controls. There may be other cofactors in addition to H. pylori that are important for the development of gastric cancer. H. pylori seems, however, to be a more important for development of gastric cancer in females than in males or males may have more confounding risk factors for gastric cancer than females.     

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.     

Detection of Helicobacter pylori in bile of cats

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.     

Detection of Helicobacter pylori in saliva and esophagus

The route of Helicobacter pylori transmission remains unclear and the currently suggested route is person-to-person transfer by faecal-oral and oral-oral mode. The aim of this study was to verify the presence of H. pylori in esophagus and saliva of humans. Saliva samples, mucosal biopsies from esophagus, gastric antrum and fundus were collected from 19 patients with positive Urea Breath Test (UBT). Gastric biopsies were used for H. pylori colture and antimicrobial susceptibility tests whereas saliva samples were collected to detect H. pylori with a Nested-PCR targeting 16S rRNA gene as well as esophagus biopsies which were also investigated with immunohistochemical staining. Helicobacter pylori was isolated in 18 patients both in gastric antrum and fundus. The molecular analysis, confirmed by comparative sequences evaluation, gave positive results in all saliva and esophageal samples whereas the immunohistochemistry revealed the presence of H. pylori in 15.8% (3/19) of the esophagus samples. Our data suggest that saliva and esophagus may be considered reservoirs for H. pylori in humans and emphasize the need to use more susceptible techniques for H. pylori detection, in particular in over-crowded sites. Identification of the transmission route of H. pylori is crucial in developing an effective plan of surveillance by finding new means of disease management.     

Antibodies against HBV, HCV and HAV detected by using microparticle enzyme immunoassay in hepatitis outpatients

The presence of the hepatitis B surface antigen (HBsAg), of the antibodies against HBc, HCV and HAV was determined in outpatients in the period September 2005 - December 2006. The serum samples were analyzed by using Enzyme Immunoassay microparticles (Abbott AxSYM System). At least one test was positive in 238 patients (15.4%) of the total of 1547 patients. Of the 238 positive subjects, in 130 positive subjects (54.6%) the existence of HBV infection could be ascertained based on the presence of HBsAg or of the antibodies against HBc or of their association; 83 patients (34.9%) presented antibodies against HCV and in other 12 patients the antibodies against HCV were associated with HBsAg or with antibodies against HBc, suggesting the coexistence of HCV and HBV infection. The antibodies against HCV and the associations between HCV and HBV were mostly detected in subjects with the diagnosis of cirrhosis, liver failure or chronic hepatitis. Of the 13 (5.46%) patients with antibodies against HAV, 6 patients presented the associations: in 2 cases antibodies anti-HAV with positive HBsAg, in 1 case antibodies anti-HAV and anti-HBc with positive HBsAg, in 2 cases antibodies anti-HAV and anti-HBc and in 1 case antibodies anti-HAV and anti-HCV.     

Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.